1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the bioactive hormone of the vitamin D endocrine system. It functions via the vitamin D receptor (VDR) to impact bone by increasing intestinal calcium absorption and by directly affecting osteoblast/osteoclast activity. The overall goal of this research proposal is to examine direct transcriptional effects of 1,25(OH)2D3 and VDR in osteoblasts. Toward this goal, expression arrays identified several novel genes that are induced by 1,25(OH)2D3 in osteoblastic cells. This proposal focuses on two 1,25(OH)2D3-induced genes that impact osteoblast biology, namely Semaphorin 3B (SEMA3B) and CCAAT/enhancer binding protein beta (C/EBPb). SEMA3B is a secreted adhesion molecule that inhibits cell growth and migration. Preliminary data show that SEMA3B is expressed in osteoblasts in vivo and is regulated by 1,25(OH)2D3 in primary osteoblasts in vitro. Imaging studies reveal a dramatic decrease in trabecular bone density of tibias obtained from a transgenic mouse expressing human SEMA3B in osteoblasts (Ob-SEMA3B). These studies are the first to address SEMA3B function in skeletal homeostasis. C/EBPb is transcription factor that is dramatically upregulated by 1,25(OH)2D3 in osteoblastic cells and it acts cooperatively with VDR to activate osteoblast gene expression. Preliminary data show that C/EBPb activates SEMA3B promoter activity in osteoblasts. A major hypothesis tested in this proposal is that VDR and C/EBPb are potent regulators of SEMA3B gene expression and of other genes that are required to maintain normal skeletal homeostasis. We propose to examine the significance of the vitamin D endocrine system in the skeletal homeostasis by focusing on the continued characterization of these new mouse models. Specifically, we propose to: 1) identify the underlying basis for the mineralization defect in the Ob- SEMA3B transgenic mouse; 2) develop an osteoblast selective knockout mouse to test the physiological role of SEMA3B in vivo; 3) examine the regulation of SEMA3B by VDR and C/EBPs at the cell and molecular levels. These studies will provide new insight into the direct actions of the vitamin D endocrine system in skeletal homeostasis. [unreadable] [unreadable] [unreadable]